CN109355269A - 鞘氨醇激酶1及其融合蛋白及其用途 - Google Patents

鞘氨醇激酶1及其融合蛋白及其用途 Download PDF

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CN109355269A
CN109355269A CN201811225720.2A CN201811225720A CN109355269A CN 109355269 A CN109355269 A CN 109355269A CN 201811225720 A CN201811225720 A CN 201811225720A CN 109355269 A CN109355269 A CN 109355269A
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段海峰
解晶
胡显文
弓景波
薛冰华
张群伟
肖秀孝
崔美兰
庞如梦
王瑞
于婷婷
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Beijing Huaao Xuande Biomedical Technology Co.,Ltd.
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Beijing Shuangyin Biotechnology Co Ltd
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Abstract

本发明提供了属于生物医药技术领域，具体涉及鞘氨醇激酶1及其融合蛋白及其用途，一种鞘氨醇激酶1或具有其活性的氨基酸序列在制备用于预防和/或治疗肥胖、高血脂症或糖尿病的蛋白质类药物中的用途。本发明还提供了一种蛋白质类药物，所述蛋白质类药物包含鞘氨醇激酶1或具有其活性的氨基酸序列。本发明还提供了上述蛋白质类药物、编码基因、表达构建体、所述的宿主细胞在制备用于预防和/或治疗肥胖、高血脂症或糖尿病的药物中的应用。本发明人发现鞘氨醇激酶1及其融合蛋白具有显著的降血糖作用和减轻体重作用，可用于制备肥胖等代谢性疾病的控制和糖尿病的蛋白质类药物。

Description

鞘氨醇激酶1及其融合蛋白及其用途
技术领域
本发明属于生物制药领域。具体地，本发明涉及鞘氨醇激酶1的用途，更具体地，本发明涉及一种鞘氨醇激酶1的融合蛋白，尤其涉及一种含有鞘氨醇激酶1和FC序列的融合蛋白及其用途。
背景技术
肥胖和2型糖尿病(T2DM)是困扰现代社会的主要公共健康问题之一。肥胖及其伴随的胰岛素抵抗是2型糖尿病发病的关键因素，据调查，80-90％的2型糖尿病患者超重或肥胖(邹大进等,上海医学,2014,37(9),729～734)。因此，有效控制血糖和体重始终是有关研究领域的焦点课题。据统计，目前全球2型糖尿病的发病人群已经达到4亿，占所有糖尿病患者的90％-95％。目前治疗糖尿病的药物主要包括胰岛素和口服降糖药如二甲双胍，但是这些药物的缺点在于容易引起低血糖，并且在对患者体重的控制等方面没有明显作用。另一类药物是GLP-1受体激动剂药物，如诺和诺德公司的利拉鲁肽，礼来公司的杜拉鲁肽等。这类药物在控制血糖的同时也有减轻体重的效果，但是主要是通过抑制患者食欲和控制患者的进食量来实现，大大降低了患者的生活质量。
在人体内还有一类在调控代谢中起到非常重要作用的酶类物质，如鞘氨醇激酶1(SphK1)等，SphK1作为新近发现的脂质激酶家族，从进化上讲在人、小鼠、酵母和植物中是保守的，该酶属于鞘脂代谢途径中的一种关键酶，催化由鞘氨醇形成鞘氨醇-1-磷酸(S1P)，是调控神经酰胺和鞘氨醇-1-磷酸(S1P)合成的“变阻器”。SphK1催化神经酰胺的代谢产物鞘氨醇生成S1P。S1P与受体结合后可调控细胞生长、凋亡、分化、造血等细胞过程。SphK1/S1P信号通路参与了多种生物学过程和疾病的发生，包括肿瘤发生和糖尿病。现有技术表明SphK1可以分泌到细胞外，但是对其在胞外的作用以及是否存在胞外的受体还不清楚(Venkataraman K,等.Biochemical Journal,2006,397(3):461-71.)。进一步地，缺失SphK1的小鼠在高糖高脂饮食下会促进胰腺细胞凋亡，从而诱发糖尿病的形成(Qi Y,等Faseb Journal,2013,27(10):4294-4304)。另外，糖尿病小鼠在注射了带有人SphK1基因的腺病毒之后，与对照组小鼠相比显示了降低的血糖和血脂水平。目前对SphK1在研究主要是基于其在细胞内的作用，因此以该蛋白为靶点开发的药物主要是使用其抗体或拮抗剂，并没有直接将该蛋白制成药物进行治疗。另一种是以病毒等为载体将SPHK1基因倒入到细胞进行基因治疗，例如用腺病毒作为载体的基因治疗，然而该方法很容易在体内产生抗药性，而治疗糖尿病等代谢性疾病需要长期用药，这就限制了该方法的使用。因此，基于人SPHK1基因进行药物研发具有广阔的前景。
发明内容
基于以上，本发明的目的是针对现有技术的不足，提供一种鞘氨醇激酶1的用途。发明人意外的发现，直接将SphK1制成蛋白质类药物，使其可以不进入细胞内，在细胞外就能发挥功能，具有显著的降血糖作用和减轻体重作用。因此，本发明提供了一种鞘氨醇激酶1在制备用于预防和/或治疗肥胖、高血脂症或糖尿病的药物中的用途。本发明还提供了一类蛋白质类药物，所述药物包含鞘氨醇激酶1(SPHK1)，本发明还提供了所述蛋白质类药物的制备方法及其用途。与现有技术相比，本发明提供的蛋白质类药物可显著降低血糖、血脂、体重和改善脂肪代谢。
一方面，本发明提供了一种鞘氨醇激酶1或具有其活性的氨基酸序列在制备用于预防和/或治疗肥胖、高血脂症或糖尿病的蛋白质类药物中的用途。
优选地，所述鞘氨醇激酶1或具有其活性的氨基酸序列包含如SEQ ID NO:1所示的氨基酸序列。
另一方面，本发明提供了一种蛋白质类药物，所述蛋白质类药物包含鞘氨醇激酶1或具有其活性的氨基酸序列；
优选地，所述蛋白质类药物为含有鞘氨醇激酶1或具有其活性的氨基酸序列的融合蛋白；更优选地，所述融合蛋白包含鞘氨醇激酶1(SPHK1)或具有其活性的氨基酸序列、FC序列和连接序列；
其中，所述FC序列选自人或动物的免疫球蛋白及其亚型和变体的氨基酸序列，或者人或动物白蛋白及其变体的氨基酸序列；
优选地，其中所述连接序列通式为(GGGGS)n，其中n为0-5的整数；优选地，n为3；
优选地，所述人或动物的免疫球蛋白选自IgG4 FC片段；更优选地，所述人或动物的免疫球蛋白选自如SEQ ID NO:12所示的氨基酸序列；
优选地，所述融合蛋白包含如SEQ ID NO:2所示的氨基酸序列。
在一个优选的实施方案中，所述融合蛋白使用聚乙二醇修饰；优选地，所述聚乙二醇的平均分子量为5-50KD；更优选地为20-45KD；优选地，所述聚乙二醇为直链或支链聚乙二醇。
另一方面，本发明提供了一种编码基因，其中，所述编码基因含有上述蛋白质类药物的编码核苷酸序列；优选地，所述编码核苷酸序列如SEQ ID NO:3所示。
再一方面，本发明提供了一种表达构建体，所述表达构建体含有上述蛋白质类药物的编码核苷酸序列；优选地，所述编码核苷酸序列如SEQ ID NO:3所示。
优选地，所述表达构建体是原核表达构建体；更优选地，所述原核表达构建体为pET载体系列；
或所述表达构建体为真核表达构建体；优选地，所述真核表达构建体为质粒DNA载体，优选pVAX1载体和pSV1.0载体；重组病毒载体，优选重组痘苗病毒载体、重组腺病毒载体或重组腺相关病毒载体；或逆转录病毒载体，优选HIV病毒载体，或慢病毒载体。
另一方面，本发明提供了一种宿主细胞，所述宿主细胞包括上述表达构建体；
优选地，当所述表达构建体是原核表达构建体时，所述宿主细胞是原核生物细胞，优选细菌细胞；或当所述表达构建体是真核表达构建体时，所述宿主细胞是真核生物细胞，优选哺乳动物细胞，更优选地为CHO细胞。
另一方面，本发明提供了一种蛋白质类药物的制备方法，所述方法包括将所述蛋白质类药物的核苷酸序列克隆至表达载体的步骤。
具体地，所述制备方法包括以下步骤：
1)构建上述蛋白质类药物的核酸序列；
2)构建包含步骤1)的核酸序列的表达载体；
3)将步骤2)的表达载体用于转染或转化宿主细胞，并使所述核酸序列在宿主细胞中表达；
4)将步骤3)中表达的蛋白进行纯化；
优选地，在步骤3)中，所述宿主细胞为CHO-S细胞。
本发明还提供了上述蛋白质类药物、编码基因、表达构建体、所述的宿主细胞在制备用于预防和/或治疗肥胖、高血脂症或糖尿病的药物组合物中的应用。
与现有技术相比，本发明具有以下优点：本发明发现鞘氨醇激酶1及其融合蛋白具有显著的降血糖作用和减轻体重作用，可用于制备肥胖等代谢性疾病的控制和糖尿病的蛋白质类药物。
附图说明
以下，结合附图来详细说明本发明的实施方案，其中：
图1为本发明pCDH-SPHK1-L-Fc的载体构建示意图；
图2为使用蛋白印迹法检测蛋白SPHK1-Fc的表达情况，其中，a是慢病毒感染细胞后，上清中蛋白的表达情况。其中“空白”是加病毒感染的细胞上清，用人IgG4Fc特异性抗体检测；b为纯化后的SDS-PAGE电泳图。
图3显示本发明的SPHK1蛋白及其融合蛋白SPHK1-Fc对II型糖尿病模型小鼠空腹血糖的影响。对照是生理盐水组。*代表与对照相比差异显著(p值<0.05)
图4显示本发明的SPHK1蛋白及其融合蛋白SPHK1-Fc治疗2周后对II型糖尿病模型小鼠体重的影响。对照是生理盐水组。*代表与对照相比差异显著(p值<0.05)
图5显示本发明的SPHK1蛋白及其融合蛋白SPHK1-Fc治疗2周后对II型糖尿病模型小鼠糖耐量的影响。对照是生理盐水组。*代表与对照相比差异显著(p值<0.05)；**代表与对照相比差异极显著(p值<0.001)；
图6显示本发明的SPHK1蛋白及其融合蛋白SPHK1-Fc治疗后对II型糖尿病模型小鼠血脂水平的影响。对照是生理盐水组。*代表与对照相比差异显著(p值<0.05)。
具体实施方式
以下参照具体的实施例来说明本发明。本领域技术人员能够理解，这些实施例仅用于说明本发明，其不以任何方式限制本发明所要保护的范围。
除非特别指明，以下实施例中所用的试剂均为分析纯级试剂，且可从正规渠道商购获得。
实施例1制备融合蛋白SPHK1-Fc
1.构建含有融合蛋白SPHK1-Fc的慢病毒表达载体pCDH-SPHK1-L-Fc
其中，鞘氨醇激酶1或具有其活性的氨基酸序列包含如SEQ ID NO:1；
MDPAGGPRGVLPRPCRVLVLLNPRGGKGKALQLFRSHVQPLLAEAEISFTLMLTERRNHARELVRSEELGRWDALVVMSGDGLMHEVVNGLMERPDWETAIQKPLCSLPAGSGNALAASLNHYAGYEQVTNEDLLTNCTLLLCRRLLSPMNLLSLHTASGLRLFSVLSLAWGFIADVDLESEKYRRLGEMRFTLGTFLRLAALRTYRGRLAYLPVGRVGSKTPASPVVVQQGPVDAHLVPLEEPVPSHWTVVPDEDFVLVLALLHSHLGSEMFAAPMGRCAAGVMHLFYVRAGVSRAMLLRLFLAMEKGRHMEYECPYLVYVPVVAFRLEPKDGKGVFAVDGELMVSEAVQGQVHPNYFWMVSGCVEPPPSWKPQQMPPPEEPL；
其编码核苷酸序列如SEQ ID NO:5所示；
ATGGACCCAGCGGGCGGCCCCCGGGGCGTGCTCCCGCGGCCCTGCCGCGTGCTGGTGCTGCTGAACCCGCGCGGCGGCAAGGGCAAGGCCTTGCAGCTCTTCCGGAGTCACGTGCAGCCCCTTTTGGCTGAGGCTGAAATCTCCTTCACGCTGATGCTCACTGAGCGGCGGAACCACGCGCGGGAGCTGGTGCGGTCGGAGGAGCTGGGCCGCTGGGACGCTCTGGTGGTCATGTCTGGAGACGGGCTGATGCACGAGGTGGTGAACGGGCTCATGGAGCGGCCTGACTGGGAGACCGCCATCCAGAAGCCCCTGTGTAGCCTCCCAGCAGGCTCTGGCAACGCGCTGGCAGCTTCCTTGAACCATTATGCTGGCTATGAGCAGGTCACCAATGAAGACCTCCTGACCAACTGCACGCTATTGCTGTGCCGCCGGCTGCTGTCACCCATGAACCTGCTGTCTCTGCACACGGCTTCGGGGCTGCGCCTCTTCTCTGTGCTCAGCCTGGCCTGGGGCTTCATTGCTGATGTGGACCTAGAGAGTGAGAAGTATCGGCGTCTGGGGGAGATGCGCTTCACTCTGGGCACCTTCCTGCGTCTGGCAGCCCTGCGCACCTACCGCGGCCGACTGGCTTACCTCCCTGTAGGAAGAGTGGGTTCCAAGACACCTGCCTCCCCCGTTGTGGTCCAGCAGGGCCCGGTAGATGCACACCTTGTGCCACTGGAGGAGCCAGTGCCCTCTCACTGGACAGTGGTGCCCGACGAGGACTTTGTGCTAGTCCTGGCACTGCTGCACTCGCACCTGGGCAGTGAGATGTTTGCTGCACCCATGGGCCGCTGTGCAGCTGGCGTCATGCATCTGTTCTACGTGCGGGCGGGAGTGTCTCGTGCCATGCTGCTGCGCCTCTTCCTGGCCATGGAGAAGGGCAGGCATATGGAGTATGAATGCCCCTACTTGGTATATGTGCCCGTGGTCGCCTTCCGCTTGGAGCCCAAGGATGGGAAAGGTGTGTTTGCAGTGGATGGGGAATTGATGGTTAGCGAGGCCGTGCAGGGCCAGGTGCACCCAAACTACTTCTGGATGGTCAGCGGTTGCGTGGAGCCCCCGCCCAGCTGGAAGCCCCAGCAGATGCCACCGCCAGAAGAGCCCTTA
所述融合蛋白SPHK1-Fc的氨基酸序列如SEQ ID NO:2所示，其核苷酸序列如SEQID NO:3所示。其N段到C段依次为SPHK1、连接序列L和Fc；
Fc的氨基酸序列如SEQ ID NO:12所示：
ESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHN AKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQ VSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQ KSLSLSPGK；
融合蛋白SPHK1-Fc的氨基酸序列如SEQ ID NO:2所示，其中，斜体加粗部分为链接序列的氨基酸序列，下划线部分为Fc的氨基酸序列：
MDPAGGPRGVLPRPCRVLVLLNPRGGKGKALQLFRSHVQPLLAEAEISFTLMLTERRNHARELVRSEELGRWDALVVMSGDGLMHEVVNGLMERPDWETAIQKPLCSLPAGSGNALAASLNHYAGYEQVTNEDLLTNCTLLLCRRLLSPMNLLSLHTASGLRLFSVLSLAWGFIADVDLESEKYRRLGEMRFTLGTFLRLAALRTYRGRLAYLPVGRVGSKTPASPVVVQQGPVDAHLVPLEEPVPSHWTVVPDEDFVLVLALLHSHLGSEMFAAPMGRCAAGVMHLFYVRAGVSRAMLLRLFLAMEKGRHMEYECPYLVYVPVVAFRLEPKDGKGVFAVDGELMVSEAVQGQVHPNYFWMVSGCVEPPPSWKPQQMPPPEEPL ESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTP EVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIE KTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSR LTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSPGK；
其核苷酸序列如SEQ ID NO:3所示其中，斜体部分为链接序列的核苷酸序列，下划线部分为Fc的核苷酸序列：
ATGGACCCAGCGGGCGGCCCCCGGGGCGTGCTCCCGCGGCCCTGCCGCGTGCTGGTGCTGCTGAACCCGCGCGGCGGCAAGGGCAAGGCCTTGCAGCTCTTCCGGAGTCACGTGCAGCCCCTTTTGGCTGAGGCTGAAATCTCCTTCACGCTGATGCTCACTGAGCGGCGGAACCACGCGCGGGAGCTGGTGCGGTCGGAGGAGCTGGGCCGCTGGGACGCTCTGGTGGTCATGTCTGGAGACGGGCTGATGCACGAGGTGGTGAACGGGCTCATGGAGCGGCCTGACTGGGAGACCGCCATCCAGAAGCCCCTGTGTAGCCTCCCAGCAGGCTCTGGCAACGCGCTGGCAGCTTCCTTGAACCATTATGCTGGCTATGAGCAGGTCACCAATGAAGACCTCCTGACCAACTGCACGCTATTGCTGTGCCGCCGGCTGCTGTCACCCATGAACCTGCTGTCTCTGCACACGGCTTCGGGGCTGCGCCTCTTCTCTGTGCTCAGCCTGGCCTGGGGCTTCATTGCTGATGTGGACCTAGAGAGTGAGAAGTATCGGCGTCTGGGGGAGATGCGCTTCACTCTGGGCACCTTCCTGCGTCTGGCAGCCCTGCGCACCTACCGCGGCCGACTGGCTTACCTCCCTGTAGGAAGAGTGGGTTCCAAGACACCTGCCTCCCCCGTTGTGGTCCAGCAGGGCCCGGTAGATGCACACCTTGTGCCACTGGAGGAGCCAGTGCCCTCTCACTGGACAGTGGTGCCCGACGAGGACTTTGTGCTAGTCCTGGCACTGCTGCACTCGCACCTGGGCAGTGAGATGTTTGCTGCACCCATGGGCCGCTGTGCAGCTGGCGTCATGCATCTGTTCTACGTGCGGGCGGGAGTGTCTCGTGCCATGCTGCTGCGCCTCTTCCTGGCCATGGAGAAGGGCAGGCATATGGAGTATGAATGCCCCTACTTGGTATATGTGCCCGTGGTCGCCTTCCGCTTGGAGCCCAAGGATGGGAAAGGTGTGTTTGCAGTGGATGGGGAATTGATGGTTAGCGAGGCCGTGCAGGGCCAGGTGCACCCAAACTACTTCTGGATGGTCAGCGGTTGCGTGGAGCCCCCGCCCAGCTGGAAGCCCCAGCAGATGCCACCGCCAGAAGAGCCCTTA GAGTCCAAATATGGTCCCCCATGCCCATCATGCCCAGCACCTGAGTTCCTGGGG GGACCATCAGTCTTCCTGTTCCCCCCAAAACCCAAGGACACTCTCATGATCTCCCGGACCCCTGAGGTCACGTGCGT GGTGGTGGACGTGAGCCAGGAAGACCCCGAGGTCCAGTTCAACTGGTACGTGGATGGCGTGGAGGTGCATAATGCCA AGACAAAGCCGCGGGAGGAGCAGTTCAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGG CTGAACGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGGCCTCCCGTCCTCCATCGAGAAAACCATCTCCAAAGC CAAAGGGCAGCCCCGAGAGCCACAGGTGTACACCCTGCCCCCATCCCAGGAGGAGATGACCAAGAACCAGGTCAGCC TGACCTGCCTGGTCAAAGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAAC TACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAGGCTAACCGTGGACAAGAGCAG GTGGCAGGAGGGGAATGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACACAGAAGAGCCTCT CCCTGTCTCCGGGTAAA
信号肽序列为IL-2信号肽序列(SP，氨基酸序列如SEQ ID NO:4所示；SEQ ID NO:4MYRMQLLSCIALSLALVTNS)
根据IL-2信号肽设计引物，包括armSP-F(上游引物)(SEQ ID NO:6：CTCCATAGAAGATTCTAGAGCTAGGGATCCGCCACCATGTACAGGATGCAACTCCTG)和armSP-R(下游引物)(SEQ ID NO:7：GGGCCGCCCGCTGGGTCCATCGAATTCGTGACAAGTGCAAG)，用质粒pFUSE-hIgG4-Fc2(购自达科为生物技术有限公司)做模板，PCR扩增片段SP(116bp)；根据SPHK1的核苷酸序列如SEQ IDNO:5设计引物，包括SPHK1-F(上游引物)(SEQ ID NO:8：ATGGACCCAGCGGGCGGCC)和SPHK1-R(下游引物)(SEQ ID NO:9：GCCACCGCCGCTTCCTCCGCCTCCGCTTCCGCCTCCGCCTAAGGGCTCTTCTGGCGGTG)用质粒pcDNA3.1-WSPK1c(军事医学科学院)做模板，PCR扩增片段SPHK1(1191bp)，
该PCR产物片段3’段含有部分连接序列L(5’-GGCGGAGGCGGAAGCGGAGGCGGAGGAAGCGGCGGTGGC-3’)；根据Fc蛋白的核苷酸序列如SEQ ID NO:3设计引物，包括Fc-F(
SEQ ID NO:10：
GCGGAGGAAGCGGCGGTGGCGGCAGCGAGTCCAAATATGGTCCCCCATGCCCATCATGC)和Fc-R(SEQ ID NO:11
GTAATCCAGAGGTTGATTGTCGACTCATTTACCCGGAGACAGGG)，用质粒pFUSE-hIgG4-Fc2(购自达科为生物技术有限公司)做模板，PCR扩增片段Fc(740bp)，该PCR产物片段5’段含有部分连接序列L(序列5’-GCGGAGGAAGCGGCGGTGGCGGCAGC-3’)。所有引物均由北京擎科新业生物技术有限公司合成。PCR反应体系和反应条件如表1和表2所示。
表1 PCR反应体系
表2：PCR反应条件
95℃ 4min
95℃ 30s
退火温度* 30s
72℃ 延伸时间<sup>#</sup>
72℃ 5min
10℃ Forever
PCR进行了30个循环。
*PCR扩增不同的片段退火温度为引物的Tm值-3℃；#PCR扩增不同的片段的延伸时间为1kb/min。
反应结束后，将所得产物进行1％琼脂糖凝胶电泳，胶回收纯化(胶回收试剂盒购自天根公司)各个PCR产物sp、SPHK1、Fc。
将质粒pCDH-CMV(购自Addgene公司)用BamHI和SalI双酶切，酶切产物切胶回收后，与上述纯化的PCR产物sp、SPHK1、Fc用无缝克隆的方法(Streamless Assembly CloningKit，购买自Clone Smarter technologies公司)连接。将连接产物转化DH5α感受态细胞(购买自天根生化科技有限公司)，转化方法参看感受态细胞说明书。将转化后菌液涂布至含有100μg/mL氨苄青霉素的LB板上，37℃过夜培养。挑取单克隆进行菌落PCR，将阳性克隆送至北京擎科新业生物技术有限公司进行测序，保存测序结果正确的克隆并提取质粒，并命名为pCDH-SPHK1-L-Fc，质粒图谱如图1所示。
实施例2制备携带质粒pCDH-SPHK1-L-Fc的慢病毒颗粒
将细胞汇合度达90％以上的293T细胞(东北农业大学Lab217胚胎工程实验室)接种到150mm培养皿中，每皿接种1.2×107个细胞，加入20ml含10％FBS的DMEM培养基，37℃、5％CO2饱和湿度培养。转染前2h，弃去原培养基，更换为18ml不含血清的DMEM培养基。将上述所制备的p-SPHK1-L-Fc质粒分别和慢病毒包装的辅助质粒pHelper1、pHelper2(东北农业大学Lab217胚胎工程实验室)按照等比率混合均匀，参照脂质体Lipofectamin 2000转染试剂盒(购自Invitrogen)说明书转染293T细胞。转染后6-8h后弃去含有转染混和物的上清，每皿加入20ml新的含5％FBS的DMEM培养基，37℃、5％CO2饱和湿度培养。24h后收集上清液保存于4℃，并加入新的20ml培养基。继续培养24h后再次收集上清液。将两次收集的上清于4℃、3500rpm离心15min，弃沉淀，将上清用Amicon Ultra-15超滤管(10KD，购自Millipore公司)离心浓缩，分别获得携带SPHK1-Fc的慢病毒颗粒。经过病毒滴度测定，将病毒颗粒稀释为1×108TU/ml，分装病毒并置于-80℃保存。
实施例3慢病毒感染CHO-S细胞及阳性单克隆的筛选和验证
3.1慢病毒感染CHO-S细胞
将悬浮FreeStyle CHO-S细胞(购买自Thermo scientific公司)以2×105细胞/mL接种于含30mL CD-SFM培养基(CD FortiCHO medium+8mM谷氨酰胺+1×HT supplement，均购自Thermo scientific公司)的125mL摇瓶中(购自康宁公司)，120rpm，8％CO2，37℃培养至对数生长期，将细胞用CD-SFM培养基稀释成4×104细胞/mL的细胞悬液。取0.5mL细胞悬液，按照感染复数(MOI)为80加入上述的慢病毒颗粒，在32℃，800g条件下离心30min。弃上清，重新加入0.5mL CD-SFM培养基重悬细胞，转移至24孔板中，在37℃，5％CO2条件下培养48-72h后用免疫印迹(Western blot)检测培养上清中目的蛋白表达情况。取30μL细胞上清经10％还原SDS-PAGE后，用低温湿法将蛋白质转印至PVDF膜上，条件为恒流300mA转膜1h。用5％脱脂牛奶/TBST溶液室温封闭1h，用偶联HRP的鼠抗人IgG4Fc抗体(1:3000稀释，购自abcam公司)检测SPHK1-Fc的表达，抗体与室温孵育1h，TBST溶液洗3次，每次10min。用ECL发光成像系统(购自北京原平皓生物技术有限公司)检测拍照。结果如图2(a)所示，在90KD处检测到特异性表达的条带，比理论分子量大，这主要是由于在CHO-S细胞表达时，对Fc上存在糖基化位点进行修饰，从而增加了分子量。
3.2阳性单克隆的筛选和验证
将感染慢病毒后的细胞用CD-SFM培养基+5％FBS重悬细胞至10个/mL，于96孔板中每孔加入100μL细胞悬液，继续培养10-14天后，显微镜下观察细胞单克隆的形成。根据人IgG ELISA定量试剂盒(购自北京达科为生物技术有限公司)的说明书，取50μL细胞单克隆培养上清进行检测蛋白表达。挑选高表达细胞株，最终每种蛋白筛选各得到2株高表达细胞株(1B7、3E8)。检测结果如表3所示。
表3单克隆细胞培养上清ELISA检测结果
编号 OD450 编号 OD450 编号 OD450
1B3 0.0854 2B10 0.3062 3C5 0.7268
1B7 1.5640 2C2 0.4588 3C6 0.9519
1C11 0.5670 2C4 0.2153 3C9 0.2938
1D6 0.2386 2E5 0.1286 3D6 0.0461
1E2 0.0237 2E9 0.0533 3E8 1.3380
1G7 0.3485 2F3 0.9846 3F2 0.3794
1G2 0.6290 2G7 0.2654 3F11 0.8542
2G11 0.1084 3G3 0.0985
实施例4 SPHK1-Fc蛋白的纯化和定量
挑选单克隆1B7至500mL摇瓶扩大培养，用1200rpm离心10min弃细胞沉淀，收集上清。上清用0.22μm滤膜过滤去除细胞碎片。用5倍柱体积的平衡缓冲液(5.6mM NaH2PO4，14.4mM Na2HPO4，0.15M NaCl，pH7.2)处理protein A亲和柱HiTrap MabSelect SuRe(购自GE通用公司)，再将上清进行上样，上样结束后，用缓冲液(5.6mM NaH2PO4·H2O，14.4mMNa2HPO4，0.5M NaCl，pH7.2)冲洗结合不牢固的杂蛋白至基线。再用洗脱液50mM柠檬酸/柠檬酸钠缓冲液(含0.02％吐温-80+5％甘露醇，pH3.2)洗脱蛋白，再用1M Tris-Cl(pH8.0)调节pH至7.0。纯化后的样品经0.22μm滤膜过滤除菌后保存于4℃。
纯化后的样品用BCA蛋白定量试剂盒(购自北京原平皓生物技术有限公司)测定蛋白的浓度。根据定量的结果，取10μg蛋白，用10％胶进行SDS-PAGE电泳，用快速蛋白染色试剂盒(购自北京原平皓生物技术有限公司)染色显影，结果扫描保存。如图2(b)所示，纯化后得到的主要蛋白大小与实施例3.1中的免疫印迹结果一致。
实施例5 SPHK1-Fc蛋白的体内药效研究。
将18只4-8周龄的雄性II型糖尿病模型鼠BKS.Cg-Dock7m+/+Leprdb/Nju(购自南京大学模式动物研究所)根据体重和空腹血糖分成3组：对照组(control，生理盐水)、给药组1(SPHK1-Fc蛋白)和给药组2(SPHK1蛋白，购自北京义翘神州生物技术有限公司，货号15679-HNCB)，每组6只小鼠。每组给药方式均为皮下注射，给药剂量均为2mg/kg。对照组和给药组1每周给药两次；给药组2每天给药一次。每周称量记录小鼠体重，并测量小鼠空腹血糖：给药当天晚上小鼠禁食12h(水正常供应)，次日早上测量血糖。根据小鼠平均血糖绘制血糖变化曲线，图3结果表明，两个给药组小鼠治疗两周后，空腹血糖明显低于对照组。图4结果表明两个给药组小鼠体重也明显轻于对照组。但是两个给药组之间在空腹血糖和体重增长上的差异都没有统计性意义。说明SPHK1和SPHK1-Fc在控制血糖和体重方面都有显著的作用。
治疗2周后检测葡萄糖耐量：测量前一晚禁食12h，测量时按照1g葡萄糖/kg的剂量腹腔注射葡萄糖，并在0，30，60和120min测量小鼠血糖。图5结果显示两个给药组比对照组在各个检测点的血糖水平都要低，而两个给药组之间的葡萄糖耐量都没有显著性差异，说明给药后小鼠对葡萄糖耐受性具有明显的改善。
葡萄糖耐量试验5天后检测血清生化指标：小鼠眼球取血，3000rpm离心10min分离血清，样品送至北京北方生科医学技术有限公司检测甘油三脂(TG)、总胆固醇(CHOL)、高密度脂蛋白(HDLC)、低密度脂蛋白(LDLC)指标。图6结果表明，SPHK1-Fc和SPHK1蛋白治疗后，小鼠的CHOL、TG和LDLC的水平明显低于对照组，说明SPHK1-Fc和SPHK1对血脂代谢具有调控作用，可以有效控制血脂水平。
[0001] 序列表
[0002] <110>北京双因生物科技有限公司 段海峰
[0003] <120>鞘氨醇激酶1及其融合蛋白及其用途
[0004] <160>12
[0005] <170>SIPOSequenceListing 1.0
[0006] <210>1
[0007] <211>384
[0008] <212>PRT
[0009] <213>Homo sapiens
[0010] <400>1
[0011] Met Asp Pro Ala Gly Gly Pro Arg Gly Val Leu Pro Arg Pro Cys Arg
[0012] 1 5 10 15
[0013] Val Leu Val Leu Leu Asn Pro Arg Gly Gly Lys Gly Lys Ala Leu Gln
[0014] 20 25 30
[0015] Leu Phe Arg Ser His Val Gln Pro Leu Leu Ala Glu Ala Glu Ile Ser
[0016] 35 40 45
[0017] Phe Thr Leu Met Leu Thr Glu Arg Arg Asn His Ala Arg Glu Leu Val
[0018] 50 55 60
[0019] Arg Ser Glu Glu Leu Gly Arg Trp Asp Ala Leu Val Val Met Ser Gly
[0020] 65 70 75 80
[0021] Asp Gly Leu Met His Glu Val Val Asn Gly Leu Met Glu Arg Pro Asp
[0022] 85 90 95
[0023] Trp Glu Thr Ala Ile Gln Lys Pro Leu Cys Ser Leu Pro Ala Gly Ser
[0024] 100 105 110
[0025] Gly Asn Ala Leu Ala Ala Ser Leu Asn His Tyr Ala Gly Tyr Glu Gln
[0026] 115 120 125
[0027] Val Thr Asn Glu Asp Leu Leu Thr Asn Cys Thr Leu Leu Leu Cys Arg
[0028] 130 135 140
[0029] Arg Leu Leu Ser Pro Met Asn Leu Leu Ser Leu His Thr Ala Ser Gly
[0030] 145 150 155 160
[0031] Leu Arg Leu Phe Ser Val Leu Ser Leu Ala Trp Gly Phe Ile Ala Asp
[0032] 165 170 175
[0033] Val Asp Leu Glu Ser Glu Lys Tyr Arg Arg Leu Gly Glu Met Arg Phe
[0034] 180 185 190
[0035] Thr Leu Gly Thr Phe Leu Arg Leu Ala Ala Leu Arg Thr Tyr Arg Gly
[0036] 195 200 205
[0037] Arg Leu Ala Tyr Leu Pro Val Gly Arg Val Gly Ser Lys Thr Pro Ala
[0038] 210 215 220
[0039] Ser Pro Val Val Val Gln Gln Gly Pro Val Asp Ala His Leu Val Pro
[0040] 225 230 235 240
[0041] Leu Glu Glu Pro Val Pro Ser His Trp Thr Val Val Pro Asp Glu Asp
[0042] 245 250 255
[0043] Phe Val Leu Val Leu Ala Leu Leu His Ser His Leu Gly Ser Glu Met
[0044] 260 265 270
[0045] Phe Ala Ala Pro Met Gly Arg Cys Ala Ala Gly Val Met His Leu Phe
[0046] 275 280 285
[0047] Tyr Val Arg Ala Gly Val Ser Arg Ala Met Leu Leu Arg Leu Phe Leu
[0048] 290 295 300
[0049] Ala Met Glu Lys Gly Arg His Met Glu Tyr Glu Cys Pro Tyr Leu Val
[0050] 305 310 315 320
[0051] Tyr Val Pro Val Val Ala Phe Arg Leu Glu Pro Lys Asp Gly Lys Gly
[0052] 325 330 335
[0053] Val Phe Ala Val Asp Gly Glu Leu Met Val Ser Glu Ala Val Gln Gly
[0054] 340 345 350
[0055] Gln Val His Pro Asn Tyr Phe Trp Met Val Ser Gly Cys Val Glu Pro
[0056] 355 360 365
[0057] Pro Pro Ser Trp Lys Pro Gln Gln Met Pro Pro Pro Glu Glu Pro Leu
[0058] 370 375 380
[0059] <210>2
[0060] <211>628
[0061] <212>PRT
[0062] <213>人工序列(Artificial Sequence)
[0063] <400>2
[0064] Met Asp Pro Ala Gly Gly Pro Arg Gly Val Leu Pro Arg Pro Cys Arg
[0065] 1 5 10 15
[0066] Val Leu Val Leu Leu Asn Pro Arg Gly Gly Lys Gly Lys Ala Leu Gln
[0067] 20 25 30
[0068] Leu Phe Arg Ser His Val Gln Pro Leu Leu Ala Glu Ala Glu Ile Ser
[0069] 35 40 45
[0070] Phe Thr Leu Met Leu Thr Glu Arg Arg Asn His Ala Arg Glu Leu Val
[0071] 50 55 60
[0072] Arg Ser Glu Glu Leu Gly Arg Trp Asp Ala Leu Val Val Met Ser Gly
[0073] 65 70 75 80
[0074] Asp Gly Leu Met His Glu Val Val Asn Gly Leu Met Glu Arg Pro Asp
[0075] 85 90 95
[0076] Trp Glu Thr Ala Ile Gln Lys Pro Leu Cys Ser Leu Pro Ala Gly Ser
[0077] 100 105 110
[0078] Gly Asn Ala Leu Ala Ala Ser Leu Asn His Tyr Ala Gly Tyr Glu Gln
[0079] 115 120 125
[0080] Val Thr Asn Glu Asp Leu Leu Thr Asn Cys Thr Leu Leu Leu Cys Arg
[0081] 130 135 140
[0082] Arg Leu Leu Ser Pro Met Asn Leu Leu Ser Leu His Thr Ala Ser Gly
[0083] 145 150 155 160
[0084] Leu Arg Leu Phe Ser Val Leu Ser Leu Ala Trp Gly Phe Ile Ala Asp
[0085] 165 170 175
[0086] Val Asp Leu Glu Ser Glu Lys Tyr Arg Arg Leu Gly Glu Met Arg Phe
[0087] 180 185 190
[0088] Thr Leu Gly Thr Phe Leu Arg Leu Ala Ala Leu Arg Thr Tyr Arg Gly
[0089] 195 200 205
[0090] Arg Leu Ala Tyr Leu Pro Val Gly Arg Val Gly Ser Lys Thr Pro Ala
[0091] 210 215 220
[0092] Ser Pro Val Val Val Gln Gln Gly Pro Val Asp Ala His Leu Val Pro
[0093] 225 230 235 240
[0094] Leu Glu Glu Pro Val Pro Ser His Trp Thr Val Val Pro Asp Glu Asp
[0095] 245 250 255
[0096] Phe Val Leu Val Leu Ala Leu Leu His Ser His Leu Gly Ser Glu Met
[0097] 260 265 270
[0098] Phe Ala Ala Pro Met Gly Arg Cys Ala Ala Gly Val Met His Leu Phe
[0099] 275 280 285
[0100] Tyr Val Arg Ala Gly Val Ser Arg Ala Met Leu Leu Arg Leu Phe Leu
[0101] 290 295 300
[0102] Ala Met Glu Lys Gly Arg His Met Glu Tyr Glu Cys Pro Tyr Leu Val
[0103] 305 310 315 320
[0104] Tyr Val Pro Val Val Ala Phe Arg Leu Glu Pro Lys Asp Gly Lys Gly
[0105] 325 330 335
[0106] Val Phe Ala Val Asp Gly Glu Leu Met Val Ser Glu Ala Val Gln Gly
[0107] 340 345 350
[0108] Gln Val His Pro Asn Tyr Phe Trp Met Val Ser Gly Cys Val Glu Pro
[0109] 355 360 365
[0110] Pro Pro Ser Trp Lys Pro Gln Gln Met Pro Pro Pro Glu Glu Pro Leu
[0111] 370 375 380
[0112] Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu
[0113] 385 390 395 400
[0114] Ser Lys Tyr Gly Pro Pro Cys Pro Ser Cys Pro Ala Pro Glu Phe Leu
[0115] 405 410 415
[0116] Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu
[0117] 420 425 430
[0118] Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser
[0119] 435 440 445
[0120] Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu
[0121] 450 455 460
[0122] Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr
[0123] 465 470 475 480
[0124] Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn
[0125] 485 490 495
[0126] Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser
[0127] 500 505 510
[0128] Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln
[0129] 515 520 525
[0130] Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val
[0131] 530 535 540
[0132] Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val
[0133] 545 550 555 560
[0134] Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro
[0135] 565 570 575
[0136] Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr
[0137] 580 585 590
[0138] Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val
[0139] 595 600 605
[0140] Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu
[0141] 610 615 620
[0142] Ser Pro Gly Lys
[0143] 625
[0144] <210>3
[0145] <211>1884
[0146] <212>DNA
[0147] <213>人工序列(Artificial Sequence)
[0148] <400>3
[0149] atggacccag cgggcggccc ccggggcgtg ctcccgcggc cctgccgcgt gctggtgctg60
[0150] ctgaacccgc gcggcggcaa gggcaaggcc ttgcagctct tccggagtca cgtgcagccc120
[0151] cttttggctg aggctgaaat ctccttcacg ctgatgctca ctgagcggcg gaaccacgcg180
[0152] cgggagctgg tgcggtcgga ggagctgggc cgctgggacg ctctggtggt catgtctgga240
[0153] gacgggctga tgcacgaggt ggtgaacggg ctcatggagc ggcctgactg ggagaccgcc300
[0154] atccagaagc ccctgtgtag cctcccagca ggctctggca acgcgctggc agcttccttg360
[0155] aaccattatg ctggctatga gcaggtcacc aatgaagacc tcctgaccaa ctgcacgcta420
[0156] ttgctgtgcc gccggctgct gtcacccatg aacctgctgt ctctgcacac ggcttcgggg480
[0157] ctgcgcctct tctctgtgct cagcctggcc tggggcttca ttgctgatgt ggacctagag540
[0158] agtgagaagt atcggcgtct gggggagatg cgcttcactc tgggcacctt cctgcgtctg600
[0159] gcagccctgc gcacctaccg cggccgactg gcttacctcc ctgtaggaag agtgggttcc660
[0160] aagacacctg cctcccccgt tgtggtccag cagggcccgg tagatgcaca ccttgtgcca720
[0161] ctggaggagc cagtgccctc tcactggaca gtggtgcccg acgaggactt tgtgctagtc780
[0162] ctggcactgc tgcactcgca cctgggcagt gagatgtttg ctgcacccat gggccgctgt840
[0163] gcagctggcg tcatgcatct gttctacgtg cgggcgggag tgtctcgtgc catgctgctg900
[0164] cgcctcttcc tggccatgga gaagggcagg catatggagt atgaatgccc ctacttggta960
[0165] tatgtgcccg tggtcgcctt ccgcttggag cccaaggatg ggaaaggtgt gtttgcagtg1020
[0166] gatggggaat tgatggttag cgaggccgtg cagggccagg tgcacccaaa ctacttctgg1080
[0167] atggtcagcg gttgcgtgga gcccccgccc agctggaagc cccagcagat gccaccgcca1140
[0168] gaagagccct taggcggagg cggaagcgga ggcggaggaa gcggcggtgg cggcagcgag1200
[0169] tccaaatatg gtcccccatg cccatcatgc ccagcacctg agttcctggg gggaccatca1260
[0170] gtcttcctgt tccccccaaa acccaaggac actctcatga tctcccggac ccctgaggtc1320
[0171] acgtgcgtgg tggtggacgt gagccaggaa gaccccgagg tccagttcaa ctggtacgtg1380
[0172] gatggcgtgg aggtgcataa tgccaagaca aagccgcggg aggagcagtt caacagcacg1440
[0173] taccgtgtgg tcagcgtcct caccgtcctg caccaggact ggctgaacgg caaggagtac1500
[0174] aagtgcaagg tctccaacaa aggcctcccg tcctccatcg agaaaaccat ctccaaagcc1560
[0175] aaagggcagc cccgagagcc acaggtgtac accctgcccc catcccagga ggagatgacc1620
[0176] aagaaccagg tcagcctgac ctgcctggtc aaaggcttct accccagcga catcgccgtg1680
[0177] gagtgggaga gcaatgggca gccggagaac aactacaaga ccacgcctcc cgtgctggac1740
[0178] tccgacggct ccttcttcct ctacagcagg ctaaccgtgg acaagagcag gtggcaggag1800
[0179] gggaatgtct tctcatgctc cgtgatgcat gaggctctgc acaaccacta cacacagaag1860
[0180] agcctctccc tgtctccggg taaa 1884
[0181] <210>4
[0182] <211>20
[0183] <212>PRT
[0184] <213>人工序列(Artificial Sequence)
[0185] <400>4
[0186] Met Tyr Arg Met Gln Leu Leu Ser Cys Ile Ala Leu Ser Leu Ala Leu
[0187] 1 5 10 15
[0188] Val Thr Asn Ser
[0189] 20
[0190] <210>5
[0191] <211>1152
[0192] <212>DNA
[0193] <213>人工序列(Artificial Sequence)
[0194] <400>5
[0195] atggacccag cgggcggccc ccggggcgtg ctcccgcggc cctgccgcgt gctggtgctg60
[0196] ctgaacccgc gcggcggcaa gggcaaggcc ttgcagctct tccggagtca cgtgcagccc120
[0197] cttttggctg aggctgaaat ctccttcacg ctgatgctca ctgagcggcg gaaccacgcg180
[0198] cgggagctgg tgcggtcgga ggagctgggc cgctgggacg ctctggtggt catgtctgga240
[0199] gacgggctga tgcacgaggt ggtgaacggg ctcatggagc ggcctgactg ggagaccgcc300
[0200] atccagaagc ccctgtgtag cctcccagca ggctctggca acgcgctggc agcttccttg360
[0201] aaccattatg ctggctatga gcaggtcacc aatgaagacc tcctgaccaa ctgcacgcta420
[0202] ttgctgtgcc gccggctgct gtcacccatg aacctgctgt ctctgcacac ggcttcgggg480
[0203] ctgcgcctct tctctgtgct cagcctggcc tggggcttca ttgctgatgt ggacctagag540
[0204] agtgagaagt atcggcgtct gggggagatg cgcttcactc tgggcacctt cctgcgtctg600
[0205] gcagccctgc gcacctaccg cggccgactg gcttacctcc ctgtaggaag agtgggttcc660
[0206] aagacacctg cctcccccgt tgtggtccag cagggcccgg tagatgcaca ccttgtgcca720
[0207] ctggaggagc cagtgccctc tcactggaca gtggtgcccg acgaggactt tgtgctagtc780
[0208] ctggcactgc tgcactcgca cctgggcagt gagatgtttg ctgcacccat gggccgctgt840
[0209] gcagctggcg tcatgcatct gttctacgtg cgggcgggag tgtctcgtgc catgctgctg900
[0210] cgcctcttcc tggccatgga gaagggcagg catatggagt atgaatgccc ctacttggta960
[0211] tatgtgcccg tggtcgcctt ccgcttggag cccaaggatg ggaaaggtgt gtttgcagtg1020
[0212] gatggggaat tgatggttag cgaggccgtg cagggccagg tgcacccaaa ctacttctgg1080
[0213] atggtcagcg gttgcgtgga gcccccgccc agctggaagc cccagcagat gccaccgcca1140
[0214] gaagagccct ta 1152
[0215] <210>6
[0216] <211>57
[0217] <212>DNA
[0218] <213>人工序列(Artificial Sequence)
[0219] <400>6
[0220] ctccatagaa gattctagag ctagggatcc gccaccatgt acaggatgca actcctg 57
[0221] <210>7
[0222] <211>41
[0223] <212>DNA
[0224] <213>人工序列(Artificial Sequence)
[0225] <400>7
[0226] gggccgcccg ctgggtccat cgaattcgtg acaagtgcaa g 41
[0227] <210>8
[0228] <211>19
[0229] <212>DNA
[0230] <213>人工序列(Artificial Sequence)
[0231] <400>8
[0232] atggacccag cgggcggcc 19
[0233] <210>9
[0234] <211>59
[0235] <212>DNA
[0236] <213>人工序列(Artificial Sequence)
[0237] <400>9
[0238] gccaccgccg cttcctccgc ctccgcttcc gcctccgcct aagggctctt ctggcggtg59
[0239] <210>10
[0240] <211>59
[0241] <212>DNA
[0242] <213>人工序列(Artificial Sequence)
[0243] <400>10
[0244] gcggaggaag cggcggtggc ggcagcgagt ccaaatatgg tcccccatgc ccatcatgc59
[0245] <210>11
[0246] <211>44
[0247] <212>DNA
[0248] <213>人工序列(Artificial Sequence)
[0249] <400>11
[0250] gtaatccaga ggttgattgt cgactcattt acccggagac aggg 44
[0251] <210>12
[0252] <211>229
[0253] <212>PRT
[0254] <213>人工序列(Artificial Sequence)
[0255] <400>12
[0256] Glu Ser Lys Tyr Gly Pro Pro Cys Pro Ser Cys Pro Ala Pro Glu Phe
[0257] 1 5 10 15
[0258] Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr
[0259] 20 25 30
[0260] Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val
[0261] 35 40 45
[0262] Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val
[0263] 50 55 60
[0264] Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser
[0265] 65 70 75 80
[0266] Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu
[0267] 85 90 95
[0268] Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser
[0269] 100 105 110
[0270] Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro
[0271] 115 120 125
[0272] Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln
[0273] 130 135 140
[0274] Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala
[0275] 145 150 155 160
[0276] Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
[0277] 165 170 175
[0278] Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu
[0279] 180 185 190
[0280] Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser
[0281] 195 200 205
[0282] Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
[0283] 210 215 220
[0284] Leu Ser Pro Gly Lys
[0285] 225

Claims (9)

1.一种鞘氨醇激酶1或具有其活性的氨基酸序列在制备用于预防和/或治疗肥胖、高血脂症或糖尿病的蛋白质类药物中的用途；
优选地，所述鞘氨醇激酶1或具有其活性的氨基酸序列包含如SEQ ID NO:1所示的氨基酸序列。
2.一种蛋白质类药物，所述蛋白质类药物包含鞘氨醇激酶1或具有其活性的氨基酸序列；
优选地，所述蛋白质类药物为含有鞘氨醇激酶1或具有其活性的氨基酸序列的融合蛋白。
3.根据权利要求2所述的蛋白质类药物，其中，所述融合蛋白包含鞘氨醇激酶1(SPHK1)或具有其活性的氨基酸序列、FC序列和连接序列；
其中，所述FC序列选自人或动物的免疫球蛋白及其亚型和变体的氨基酸序列，或者人或动物白蛋白及其变体的氨基酸序列；
优选地，所述连接序列通式为(GGGGS)n，其中n为0-5的整数；更优选地，n为3；
优选地，所述人或动物的免疫球蛋白选自IgG4FC片段；更优选地，所述人或动物的免疫球蛋白选自如SEQ ID NO:12所示的氨基酸序列；
优选地，所述融合蛋白包含如SEQ ID NO:2所示的氨基酸序列。
4.如权利要求2所述的的蛋白质类药物，其中，所述融合蛋白使用聚乙二醇修饰；优选地，所述聚乙二醇的平均分子量为5-50KD；更优选地为20-45KD；进一步优选地，所述聚乙二醇为直链或支链聚乙二醇。
5.一种编码基因，其中，所述编码基因含有如权利要求2-4中任一项所述的蛋白质类药物的编码核苷酸序列；优选地，所述编码核苷酸序列如SEQ ID NO:3所示。
6.一种表达构建体，所述表达构建体含有如权利要求2-4中任一项所述的蛋白质类药物的编码核苷酸序列；优选地，所述编码核苷酸序列如SEQ ID NO:3所示；
优选地，所述表达构建体是原核表达构建体；更优选地，所述原核表达构建体为pET载体系列；
或所述表达构建体为真核表达构建体；优选地，所述真核表达构建体为质粒DNA载体，优选pVAX1载体和pSV1.0载体；重组病毒载体，优选重组痘苗病毒载体、重组腺病毒载体或重组腺相关病毒载体；或逆转录病毒载体，优选HIV病毒载体，或慢病毒载体。
7.一种宿主细胞，所述宿主细胞包含如权利要求6所述的表达构建体；
优选地，当所述表达构建体是原核表达构建体时，所述宿主细胞是原核生物细胞，优选细菌细胞；或当所述表达构建体是真核表达构建体时，所述宿主细胞是真核生物细胞，优选哺乳动物细胞，更优选地为CHO细胞。
8.一种蛋白质类药物的制备方法，所述方法包括将如权利要求2-4中任一项所述的蛋白质类药物的核苷酸序列克隆至表达载体的步骤。
具体地，所述制备方法包括以下步骤：
1)构建上述蛋白质类药物的核酸序列；
2)构建包含步骤1)的核酸序列的表达载体；
3)将步骤2)的表达载体用于转染或转化宿主细胞，并使所述核酸序列在宿主细胞中表达；
4)将步骤3)中表达的蛋白进行纯化；
优选地，在步骤3)中，所述宿主细胞为CHO-S细胞。
9.如权利要求2-4中任一项所述的蛋白质类药物、如权利要求5所述的编码基因、如权利要求6所述的表达构建体、如权利要求7所述的宿主细胞在制备用于预防和/或治疗肥胖、高血脂症或糖尿病的药物组合物中的应用。
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